Keratinocytes comprise the principal cell type in stratified squamous ephithelia (e.g., epidermis), major sites of chronic human disease. This proposal concerns several aspects of cross-linked envelope formation, a distinctive property of this cell type, in human and rat keratinocytes serially cultivated with 3T3 feeder layer support. First, the biochemical basis of envelope formation will be explored in light of our finding that considerable transglutaminase activity is membrane bound. The interaction of this enzyme with involucrin and other possible envelope constituents in membrane fractions will be explored to ascertain the mechanism of envelope cross-linking at the cell periphery. Second, our observation that culture conditions strongly modulate envelope forming ability will be extended, with emphasis on the actions of corticosteroids, retinoids and an unidentified serum factor. This factor will be characterized and, if possible, identified. We have found that keratinocytes grown under altered culture conditions or from different epithelia are distinguishable not only by envelope forming ability but also by toxic responses to 3-methylcholanthrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin. A third aspect of the proposal is to examine in a systematic fashion cellular responses to these and other model toxic agents (PCBs, nitrosamines) in keratinocytes which exhibit intrinsic differences in regulation of envelope formation. The long range objectives of this work are 1) to provide a system for examining in keratinocytes the mechanisms of corticosteroid and retinoid action and effectiveness of pharmaceutical derivatives, 2) to improve human risk assessment in chemical testing, and 3) to provide a better understanding of keratinocyte physiology and intrinsic divergence, with special relevance to squamous metaplasia. In addition to culture studies, we propose to apply our method of immunoperoxidase staining of involucrin in paraffin embedded tissue sections to regions of squamous metaplasia and leukoplakia. This work may aid in recognition and differential diagnosis of neoplastic and perhaps other human keratinocyte lesions.